CN108828215A - A kind of glutathione reductase assay kit and its preparation method and application - Google Patents
A kind of glutathione reductase assay kit and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of glutathione reductase assay kit, and kit contains reagent R1 and reagent R2；Contain following component in reagent R1：Tris buffer 50-200mmol/L, EDTA 0.5-2mmol/L, pyruvate carboxylase 1-5KU/L, ascorbic acid oxidase 1-5KU/L, potassium ferrocyanide 5-20mg/L, surfactant, preservative；Contain following component in reagent R2：Tris buffer 50-200mmol/L, EDTA 0.5-2mmol/L, GSSG 2-10mmol/L, NADPH 0.1-0.5 mmol/L, stabilizer 1-15g/L, preservative 0.5-2g/L.The kit is that a kind of stability is strong, the liquid reagent box of strong antijamming capability.The present invention discloses preparation methods and application.
Description
Technical field
The present invention relates to biochemical reagents determination techniques field, in particular to a kind of glutathione reductase assay kit,
Further relate to the preparation method and application of the glutathione reductase assay kit.
Background technique
Glutathione reductase (GR) is a kind of flavo-enzyme, and per molecule zymoprotein contains the FAD of a molecule.By coenzyme
NADPH hydrogen supply, catalysis oxidation type glutathione (GSSG) are reduced into reduced glutathione (GSH).Reduced glutathione
(GSH) enzyme containing sulfydryl (- SH) can be made to be in reducing condition and activated state, the integrality of erythrocyte membrane is maintained, prevent blood red
Protein oxidation.GR is strong and weak in machine activity in vivo, is followed successively by liver, kidney, pancreas, the heart, thyroid gland, red blood cell and blood plasma.GR is positioned at micro-
Plastochondria and cytosol fractions.Because the histocyte of each internal organs generally contains GR, thus GR rises in body redox reaction
Very important status.
In normal body metabolic process, body is there are effective Antioxidative Defense System, the oxygen radical that constantly generates
It is constantly removed under the action of antioxidant system, to maintain the concentration of physiological level.Antioxidant system includes intracellular
The antioxidant of Antioxidant Enzyme Systems and non-enzymatic, wherein most importantly glutathione antioxidant object enzyme system.Glutathione
Polyphenoils enzyme system mainly includes glutathione (GSH), glutathione peroxidase (GPX), glutathione reductase
(GR), glutathione sulfydryl transferase (GST).Glutathione is synthesized naturally in human cell, is mainly synthesized in liver, by
The small molecule tripeptide compound that glutamic acid, cysteine, glycine form is intracellular main non-protein sulfhydryl chemical combination
Object.Exist in the cell under normal environment with thiol reduction (GSH), reduced form is its main activated state, is accounted about
95%；Oxidized form of glutathione (GSSG) is inactive state, accounts for about 1%.Endogenous GSH is primarily present in endochylema, by γ-
Glutamylcysteine ??synthase and glutathione synthetase catalyze and synthesize.The sulfydryl that GSH contains is that it plays main function
Group.Many vital movements include gene expression regulation, enzymatic activity and Metabolism regulation, the protection to cell, amino acid transport,
Immunity regulation etc. plays direct or indirect effect.Oxidative stress or electrophilic compound attack can make intracellular GSH content
Decline, or it is made to be changed into double sulphur oxidisability (GSSG).GR is a kind of flavoprotein oxidoreducing enzyme, is with Reducing Coenzyme II
Hydrogen donor can be catalyzed GSSG and be reduced into GSH, and for maintaining the stabilization of organism GSH and GSSG ratio, oxygen radical is flat in keeping body
Weighing apparatus plays an important role.GPX can be special catalysis GSH to the reduction reaction of hydrogen peroxide, play protection membrane structure and
Fully functional effect, and lipid peroxide can be made to become nontoxic hydroxylate.Condition GST low in GPX vigor
Under, have the function of removing internal oxygen radical, while can make harmful electrophilic substance with GSH in conjunction with, discharge it in vitro, from
And play the function of protecting vivo protein and nucleic acid etc..
The method of domestic measurement glutathione reductase mainly has Elisa method and ultraviolet enzyme process at present, the former needs craft
Operation, takes a long time, step is comparatively laborious, expensive.The method that the country mainly promotes at present is ultraviolet enzyme process, but existing
The kit of ultraviolet enzyme process glutathione reductase is mainly import powdered reagent, expensive although result is accurate, is made
It is inconvenient for operation with preceding needing to redissolve.Need to use as early as possible after redissolution, using it is very inconvenient and redissolve after use endless meeting
There is a large amount of waste.And the domestic liquid double reagent that most convenient uses on clinical biochemical analyzer is presently, there are stability is poor,
The disadvantages of poor anti jamming capability.
Summary of the invention
To solve the above-mentioned problems, the present invention provide a kind of glutathione reductase assay kit and preparation method thereof and
Using the kit is that a kind of stability is strong, the liquid reagent box of strong antijamming capability.
The present invention is achieved by the following technical solutions：
A kind of glutathione reductase assay kit, kit contain reagent R1 and reagent R2；
Contain following component in reagent R1：Tris buffer 50-200mmol/L, EDTA 0.5-2mmol/L, pyruvic acid carboxylic
Change enzyme 1-5KU/L, ascorbic acid oxidase 1-5KU/L, potassium ferrocyanide 5-20mg/L, surfactant 1-5ml/L, preservative
0.5-2g/L；
Contain following component in reagent R2：Tris buffer 50-200mmol/L, EDTA 0.5-2mmol/L, GSSG2-
10mmol/L, NADPH 0.1-0.5mmol/L, stabilizer 1-15g/L, preservative 0.5-2g/L.
Preferably, the pH value of reagent R1 is 6.5-7.5；The pH value of reagent R2 is 9.0-10.0.
Preferably, surfactant described in reagent R1 is polysorbas20, TritonX-100, TritonX-405,12
One of sodium alkyl sulfate and Brij35 are a variety of.
Preferably, stabilizer described in reagent R2 is the mixture of soluble starch, trehalose, glycerol, and solubility is formed sediment
Powder, trehalose, glycerol mass ratio 2：1-3:6-10.
Preferably, preservative described in reagent R1 be one of Sodium azide, PC300, MIT and gentamicin sulphate or
It is a variety of.
Preferably, preservative described in reagent R2 be one of Sodium azide, PC300, MIT and gentamicin sulphate or
It is a variety of.
Preferably, the volume ratio of the reagent R1 and reagent R2 is 1~5:1.
It is highly preferred that the volume ratio of the reagent R1 and reagent R2 is 4:1.
The preparation method of glutathione reductase assay kit described above, includes the following steps：
(1) preparation of reagent R1：Suitable quantity of water is taken, raw material shown in R1 is sequentially added into, after stirring evenly one raw material of dissolution
Next raw material is added, is adjusted with hydrochloric acid or sodium hydroxide to pH value 6.5-7.5, constant volume to required volume；
(2) preparation of reagent R2：Suitable quantity of water is taken, raw material shown in R1 is sequentially added into, after stirring evenly one raw material of dissolution
Next raw material is added, is adjusted with hydrochloric acid or sodium hydroxide to pH value 9-10, constant volume to required volume.
The application of glutathione reductase assay kit described above, the diagnosing and treating purpose for non-disease are surveyed
Determine the concentration of serum Glutathione fabk polypeptide.
Glutathione reductase assay kit principle of the present invention is glutathione reductase (GR) catalysis oxidation type paddy Guang
Sweet peptide (GSSG) is reduced into reduced glutathione (GSH), while reduced Coenzyme II (NADPH) is oxidized into codehydrogenase Ⅱ
(NADP+).NADPH has specificabsorption peak in wavelength 340nm, and the rate of oxidation is directly proportional to the activity of GR in serum,
The rate that the decline of NADPH absorbance is measured at 340nm can calculate GR activity.
The reaction equation of reaction principle is as follows:
Beneficial effects of the present invention：
1. the stable glutathione reductase assay kit of the present invention is liquid double reagent, prepared without redissolving, corkage
It can directly use.
2. the present invention by adding pyruvate carboxylase, ascorbic acid oxidase, potassium ferrocyanide in reagent R1, can incite somebody to action
Interfering substance (such as pyruvic acid, ascorbic acid, bilirubin) removal in serum, improves the accuracy of measurement.
3. the present invention can protect reagent R2 by the mixture of addition soluble starch, trehalose, glycerol in reagent R2
In glutathione, so that guaranteeing that the corkage of reagent room temperature is kept in dark place stablizes 30 days, 2-8 DEG C is closed bottle preservation and stablized 12 months, complete
The needs of full up foot clinical examination.
Detailed description of the invention
Fig. 1 is the thermostabilization of 1 glutathione reductase assay kit of the embodiment of the present invention and 4,5,6 kit of comparative example
Property variation.
Specific embodiment
The following is specific embodiments of the present invention is described with reference to the drawings, and further retouches to technical solution of the present invention work
It states, however, the present invention is not limited to these examples.
The test condition of kit measurement serum Glutathione fabk polypeptide of the present invention is：Method：Performance rate method；Master/slave wave
It is long：340nm/405nm；Temperature：37℃；Correct type：Linearly；Calibration method：Two-point calibration；The Direction of Reaction：Downwards.
Concrete operations are as shown in table 1.
1 glutathione reductase of table measures reagent operation step
Calculated result：
Sample requirement：
1. not haemolysis serum.
2. Almost Sure Sample Stability：2~8 DEG C of sample preservations can stablize 3 days, and -20 DEG C of preservations can stablize 2 weeks.
Embodiment 1
The group of reagent R1 is divided into：Tris buffer 100mmol/L, EDTA 0.5mmol/L, pyruvate carboxylase 1.5KU/
L, ascorbic acid oxidase 2KU/L, potassium ferrocyanide 6mg/L, polysorbas20 1ml/L, Sodium azide 0.5g/L；
Group in reagent R2 is divided into：Tris buffer 100mmol/L, EDTA 0.5mmol/L, GSSG 2mmol/L,
NADPH0.2mmol/L, soluble starch 1g/L, trehalose 1g/L, glycerol 4g/L, Sodium azide 0.5g/L.
The pH value of reagent R1 is 7.0；The pH value of reagent R2 is 9.5；
Preparation method:(1) preparation of reagent R1：Suitable quantity of water is taken, raw material shown in R1 is separately added into, stirs evenly one original of dissolution
Next raw material is added after material, is adjusted with hydrochloric acid or sodium hydroxide to required pH value, constant volume to required volume.(2) reagent R2
It prepares：Take suitable quantity of water, be separately added into raw material shown in R1, next raw material is added after stirring evenly one raw material of dissolution, with hydrochloric acid or
Sodium hydroxide is adjusted to required pH value, constant volume to required volume.
Embodiment 2
The group of reagent R1 is divided into：Tris buffer 80mmol/L, EDTA 0.8mmol/L, pyruvate carboxylase 1.2KU/L,
Ascorbic acid oxidase 2.5KU/L, potassium ferrocyanide 8mg/L, brij35 1ml/L, Sodium azide 0.5g/L；
Group in reagent R2 is divided into：Tris buffer 80mmol/L, EDTA 0.8mmol/L, GSSG 2mmol/L, NADPH
0.2mmol/L, soluble starch 1.5g/L, trehalose 1.5g/L, glycerol 6g/L, Sodium azide 0.5g/L.
The pH value of reagent R1 is 6.5；The pH value of reagent R2 is 9.3；
The preparation method is the same as that of Example 1 for 2 kit of embodiment
Embodiment 3
The group of reagent R1 is divided into：Tris buffer 150mmol/L, EDTA 1mmol/L, pyruvate carboxylase 2KU/L, resist
Bad hematic acid oxidizing ferment 3KU/L, potassium ferrocyanide 10mg/L, TritonX-100 1ml/L, Sodium azide 0.5g/L；
Group in reagent R2 is divided into：Tris buffer 150mmol/L, EDTA 1mmol/L, GSSG 2mmol/L, NADPH
0.2mmol/L, soluble starch 2g/L, trehalose 2g/L, glycerol 9g/L, Sodium azide 0.5g/L.
The pH value of reagent R1 is 7.0；The pH value of reagent R2 is 9.0；
The preparation method is the same as that of Example 1 for 3 kit of embodiment
Comparative example 1
Commercially available import glutathione reductase assay kit.
Comparative example 2
Difference with 1 Glutathione fabk polypeptide assay kit of embodiment is only that in reagent R1 without carboxylase
Enzyme, other are same as Example 1, are not repeated herein.
Comparative example 3
Difference with 1 Glutathione fabk polypeptide assay kit of embodiment is only that in reagent R1 without ascorbic acid oxygen
Change enzyme, other are same as Example 1, are not repeated herein.
Comparative example 4
Difference with 1 Glutathione fabk polypeptide assay kit of embodiment is only that in reagent R1 without ferrocyanide
Potassium, other are same as Example 1, are not repeated herein.
Comparative example 5
Difference with 1 Glutathione fabk polypeptide assay kit of embodiment is only that in reagent R2 forms sediment without soluble
Powder, other are same as Example 1, are not repeated herein.
Comparative example 6
Difference with 1 Glutathione fabk polypeptide assay kit of embodiment, which is only that in reagent R2, is free of trehalose,
He is same as Example 1, is not repeated herein.
Comparative example 7
Difference with 1 Glutathione fabk polypeptide assay kit of embodiment, which is only that in reagent R2, is free of glycerol, other
It is same as Example 1, it is not repeated herein.
Performance detection
1, accuracy
The source of people blood serum sample for taking 40 parts of various concentrations within the scope of detectable concentration, using to embodiment 1 as compare reagent,
40 samples are measured respectively, and each sample measures are primary.Correlation analysis is carried out to two groups of testing results, calculates correlation coefficient r；
To compare 1 testing result of embodiment as target value, the relative deviation (Bias%) of 40 pairs of data is calculated separately.It is required that r is not less than
0.990, relative deviation is no more than ± 10%.It is shown in Table 2, table 3.
2 embodiment 1 of table, comparative example 2, comparative example 3, comparative example 4 are opposite with comparative example 1 respectively
Deviation
3 embodiment 1 of table, comparative example 2, comparative example 3, comparative example 4 phase with comparative example 1 respectively
Relationship number
Conclusion：It was found from table 2 and table 3：The relative deviation maximum value of embodiment 1 and comparative example 1 is 2.97%, phase relation
Number r is 0.9979, illustrates the reagent strong antijamming capability of embodiment 1, all relatively more accurate for various concentration pattern detection result.
And comparative example 2, comparative example 3, comparative example 4 and comparative example 1 relative deviation some be more than ± 20%, phase relation
Number both less than 0.990, main cause is in serum sample there are interfering substance, and content is different in different samples, affects detection
As a result accuracy.Pyruvate carboxylase, ascorbic acid oxidase, potassium ferrocyanide are added in reagent produced by the present invention,
This can utmostly remove the interfering substance in serum sample, improve the accuracy of clinical examination, ensure that testing result
Reliability.
2, Detection of Stability：
By (this is sentenced for embodiment 1, or the other embodiment of the present invention) of the embodiment of the present invention and comparative example
5, the reagent in 6,7 is put into togerther in 37 DEG C of water baths, is detected the quality-control product that target value is 60.5 ± 6U/L daily, is monitored quality-control product
Variation.It is shown in Table 4
4 embodiment 1 of table and comparative example 5, embodiment 6, the variation of the quality-control product concentration of embodiment 7.
Conclusion：From table 4 and Fig. 1 it is found that the stabilization of kit of embodiment 1 is better than 5,6,7 kit of comparative example
Stability illustrates the present invention while soluble starch, trehalose, glycerol, the good collaboration played to reagent stability is added
Effect, can significantly improve the stability of glutathione reductase assay kit.
Specific embodiment described herein is only an example for the spirit of the invention.The neck of technology belonging to the present invention
The technical staff in domain can do various modifications or supplement or is substituted in a similar manner to described specific embodiment, but simultaneously
Spirit or beyond the scope defined by the appended claims of the invention is not deviated by.
It is skilled to this field although present invention has been described in detail and some specific embodiments have been cited
For technical staff, as long as it is obvious for can making various changes or correct without departing from the spirit and scope of the present invention.
Claims (10)
1. a kind of glutathione reductase assay kit, which is characterized in that kit contains reagent R1 and reagent R2；
Contain following component in reagent R1：Tris buffer 50-200mmol/L, EDTA 0.5-2mmol/L, carboxylase
Enzyme 1-5KU/L, ascorbic acid oxidase 1-5KU/L, potassium ferrocyanide 5-20mg/L, surfactant 1-5ml/L, preservative
0.5-2g/L；
Contain following component in reagent R2：Tris buffer 50-200mmol/L, EDTA 0.5-2mmol/L, GSSG 2-
10mmol/L, NADPH 0.1-0.5 mmol/L, stabilizer 1-15g/L, preservative 0.5-2g/L.
2. glutathione reductase assay kit according to claim 1, which is characterized in that the pH value of reagent R1 is
6.5-7.5；The pH value of reagent R2 is 9.0-10.0.
3. glutathione reductase assay kit according to claim 1, which is characterized in that table described in reagent R1
Face activating agent is one of polysorbas20, TritonX-100, TritonX-405, lauryl sodium sulfate and Brij35 or more
Kind.
4. glutathione reductase assay kit according to claim 1, which is characterized in that steady described in reagent R2
Determine agent be soluble starch, trehalose, glycerol mixture, soluble starch, trehalose, glycerol mass ratio 2：1-3:6-
10。
5. glutathione reductase assay kit according to claim 1, which is characterized in that prevent described in reagent R1
Rotten agent is one of Sodium azide, PC300, MIT and gentamicin sulphate or a variety of.
6. glutathione reductase assay kit according to claim 1, which is characterized in that prevent described in reagent R2
Rotten agent is one of Sodium azide, PC300, MIT and gentamicin sulphate or a variety of.
7. glutathione reductase assay kit according to claim 1, which is characterized in that the reagent R1 and reagent
The volume ratio of R2 is 1 ~ 5:1.
8. glutathione reductase assay kit according to claim 7, which is characterized in that the reagent R1 and reagent
The volume ratio of R2 is 4:1.
9. the preparation method of glutathione reductase assay kit described in a kind of one of claim 1-8, which is characterized in that
Include the following steps：
（1）The preparation of reagent R1：Suitable quantity of water is taken, raw material shown in R1 is sequentially added into, is added after stirring evenly one raw material of dissolution
Next raw material is adjusted with hydrochloric acid or sodium hydroxide to pH value 6.5-7.5, constant volume to required volume；
（2）The preparation of reagent R2：Suitable quantity of water is taken, raw material shown in R1 is sequentially added into, is added after stirring evenly one raw material of dissolution
Next raw material is adjusted with hydrochloric acid or sodium hydroxide to pH value 9.0-10.0, constant volume to required volume.
10. the application of glutathione reductase assay kit described in a kind of one of claim 1-8, for examining for non-disease
The concentration of disconnected and therapeutic purposes measurement serum Glutathione fabk polypeptide.
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| CN110734952A (en) * | 2019-11-01 | 2020-01-31 | 江西乐成生物医疗有限公司 | Glutathione reductase detection kit and application |
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